Various tissue culture procedures for propagating hepatitis A virus have been disclosed, such as, for example, U.S. Pat. No. 4,164,566; European Patent Application No. 25,745; Frosner, et al, Propagation of Human Hepatitis A virus in a Hepatoma Cell Line, Infection, 6: 303-306; Flehmig, Hepatitis A-virus in cell culture: I. Propagation of Different Hepatitis Virus Isolates in a Fetal Rhesus Monkey Kidney Cell Line (Rfnk-4), Med. Microbiol. Immunol., 168: 239-248; and Daemer, et al, Propagation of Human Hepatitis A Virus in African Green Monkey Kidney Cell Culture: Primary and Serial Passage, Infection and Immunity, 32: 388-393. However, such methods are hereinafter referred to as "acute infection" procedures wherein multiple passages of cell free virus in tissue cultures are required before an acceptable virus titer is obtained. In accordance with acute infection procedures, a cell tissue culture is infected with a specimen containing hepatitis A virus. The infected cell culture is grown in an appropriate medium through "confluency" and then the fully grown cells are allowed to "age". The "aged" cells are " killed" and the virus is extracted. The extracted cell free virus is subsequently utilized to reinfect a new cell culture. This process is repeated several times, generally referred to as passages of the virus, until an acceptable virus titer is obtained. However, "acute infection" or so-called "reinfection" procedures are generally time consuming and inefficient in that such procedures generally involve termination of the infected cultures and infection of new cultures in order to obtain a suitable virus titer. As a result, material, manpower, and physical space requirements for tissue culture procedures utilizing acute infection techniques are high. Furthermore, some disclosed acute infection procedures are "indirect", that is, require repeated adaptation of the virus in marmosets before propagation in tissue cultures.
It is an object of the present invention to provide a tissue culture procedure for the production of hepatitis A virus in vitro, which eliminates the tedious and time consuming acute infection procedures requiring "reinfection" or repeated passages of cell free virus. Also, it is a further object of the present invention to provide a procedure for increasing the yields of hepatitis A virus obtained from a tissue culture procedure.